Project Summary The goal of our research is to understand the cellular and molecular mechanisms of mast cell-mediated itch and how mast cells interact with sensory nerves to induce itch. Primary sensory neurons in dorsal root ganglia (DRG) play an essential role in generating itch. We showed that several Mrgprs, a large family of G protein-coupled receptors (GPCRs) are specifically expressed in the DRG and function as novel itch receptors by detecting various pruritogens. Although sensory neurons are pivotal in facilitating itch sensation, other cell types in the skin such as mast cells and keratinocytes also play a major role in generating itch. Mast cells are innate immune cells embedded in most tissues of the body and secrete a wide range of substances such as histamine and serotonin. Inappropriate mast cell activation has been linked to an increasing number of serious diseases, including skin diseases, chronic itch, gastrointestinal disorders, and asthma. Besides the classical IgE-dependent pathway, a variety of cationic substances, collectively called basic secretagogues, can also activate mast cells. Recently we identified that MrgprB2, the orthologue of human MrgprX2, is the mast cell receptor for basic secretagogues in mice. Unlike Mrgprs found in sensory neurons, both MrgprB2 and MrgprX2 are exclusively expressed in mast cells and activated by various basic secretagogues (e.g. compound 48/80 and substance P). Although mast cells have been implicated in many itch associated skin diseases, the underlying cellular and molecular mechanisms have not been fully characterized. In this proposal, we will take molecular, genetic, behavioral, and imaging approaches to dissect the functions of MrgprB2 and properties of Mrgpr-expressing mast cells in itch. Our preliminary data showed that skin anti-microbial peptide PAMP, which induces itch in humans, is an MrgprB2/X2 ligand. We also found that PAMP-induced mast cell activation and itch are mediated by MrgprB2. In Aim I, we will determine whether MrgprB2 is involved in acute and chronic itch. Furthermore, we will test the hypothesis that PAMP and substance P are candidates for the endogenous ligands of MrgprB2 in itch. Finally we will examine what mediators are released from mast cells activated by MrgprB2 in itch. Since MrgprB2 is expressed in ~100% of mast cells, it provides us with a great genetic tool to specifically study mast cells. In Aim II, we will study how mast cells contribute to acute and chronic itch by MrgprB2-Cre dependent genetic manipulation of mast cells (i.e. labeling, ablation, and activation). Using Pirt- GCaMP3 mice we successfully imaged DRG neuron activation in response to pruritogens at a populational level in live mice. In Aim III, we will use this powerful in vivo DRG imaging technique to answer important questions on itch: whether activation of mast cells can activate sensory neurons under acute and chronic itch conditions, what types of sensory neurons, and what mediators are involved. The results of the project will provide insight into key itch mechanisms and open the door for the development of novel itch therapeutics.